These results also suggest that Mel-18

can function as conventional transcriptional repressor in Th cells in a gene-dependent Pifithrin-�� solubility dmso context. We did not notice any changes in the expression levels of Ifng or Il4 mRNAs as a result of Mel-18 or Ezh2 knockdown in Th17 cells (Fig. 2G and H). We neither found any changes in the expression levels of the two Gata3 transcripts 71 (data not shown). The mRNA level of Tbx21, encoding T-bet, was increased in some experiments following Ezh2 knockdown (data not shown), but this result was inconsistent. In summary, our results show that PcG proteins positively regulate the expression of Il17a, Il17f and Rorc in restimulated Th17 cells. Considering the binding pattern of PcG proteins at the promoter of Il17a, a direct transcriptional regulation is suggested, but the involvement of additional indirect regulatory pathways is

also possible. The inducible binding activity of Mel-18 and Ezh2 at the Il17a promoter was regulated by factors downstream to the TCR (Fig. 1). However, since PcG proteins are expressed non-differentially in Th1, Th2 and R788 price Th17 cells (here and 66), the lineage selectivity of their binding pattern is probably instructed by the polarizing cytokines. We aimed therefore to determine whether the presence of the polarizing cytokines is required for the binding activity of PcG proteins at the Il17a promoter in differentiated Th17 cells. First, we wanted to examine the requirement of these cytokines to maintain Th17 phenotype under our experimental conditions. Freshly purified CD4+ T cells were differentiated under Th17 conditions

3-oxoacyl-(acyl-carrier-protein) reductase for 6 days (TGF-β and IL-6 including IL-23) and then were restimulated with PMA and ionomycin for 2 h in either the presence of Th17 skewing cytokines, without cytokines or in the presence of the Th1 polarizing cytokine IL-12 (data not shown). We did not observe statistically significant changes in the expression levels of the mRNAs of Rorc, Rora, Il17a and Il17f. Similar results were observed when the cells were restimulated 2 h with anti-CD3 and anti-CD28 antibodies (data not shown). Therefore, shortly after restimulation Th17 cells maintain their ability to express the specific cytokines and transcription factors in the absence of polarizing cytokines. Next we wanted to determine whether a continuous presence of the polarizing cytokines is necessary to maintain the Th17 transcriptional program during a longer restimulation. Freshly purified CD4+ T cells were differentiated with TGF-β, IL-6 and IL-23 for 6 days and then were restimulated with anti-CD3 and anti-CD28 antibodies for 18 h in the presence of different cytokines as indicated in Fig. 3A.